Purification, Ligation and Cyclization of Recombinant Systems... · Purification, Ligation and Cyclization

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  • Version 1.211/00

    Catalog #E6950SStore at 20CNote: Store chitinbeads at 4C

    BioLabsNEW ENGLAND

    Inc.

    IMPACT-TWIN

    Purification, Ligation and Cyclizationof Recombinant Proteins

    I n s t r u c t i o n M a n u a l

    For additional information, including vector sequences andfrequently asked questions, see the NEB website: www.neb.com

  • Table of Contents:Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3System Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4pTWIN Vector Map . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6Protein Purification Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

    N-terminal Fusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11C-terminal Fusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15N- and C-terminal Intein Fusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

    Cloning into a pTWIN Vector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27Affinity Purification and On-column Cleavage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30Simplified Purification Protocol for Test Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34Media and Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38Frequently Asked Questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46Appendices

    Appendix I: Comparison Between Intein Tag Fusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48Appendix II: Protein Labeling and Intein-mediated Protein Ligation . . . . . . . . . . . . . . . . . . . 50Appendix III: Sequencing Primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52Appendix IV: The Chemical Mechanism of Protein Splicing . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54Appendix V: The Chemical Mechanism of Thiol-inducible and pH-inducible Cleavage

    Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56Appendix VI: Regeneration of Chitin Resin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58Appendix VII: Research Use Assurance Statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

    Kit Components Sold Separately/Companion Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

    Notice to Buyer/User: The buyer and user have a non-exclusive license to use this system or any components thereof forRESEARCH PURPOSES ONLY. See RESEARCH USE ASSURANCE STATEMENT attached hereto as Appendix VII for detail on termsof the license granted hereunder.Information presented herein is accurate and reliable to the best of our knowledge and belief, but is not guaranteed to be so.Nothing herein is to be construed as recommending any practice or any product in violation of any patent or violation of any lawor regulation. It is the users responsibility to determine for himself or herself the suitability of any material and/or procedure for aspecific purpose and to adopt such safety precautions as may be necessary.

    1

  • The System Includes:IMPACT-TWIN System components are described in detail on page 4. A list ofcomponents sold separately and companion products can be found on page 60.

    Vector DNA (3 vectors)10 g of each (50 l)

    Sequencing Primers (3 primers)200 picomoles of each

    E. coli Strain ER25660.2 ml stock cells

    Anti-Chitin Binding Domain Serum (rabbit)50 l

    Chitin Beads (store at 4C)20 ml

    1, 4-Dithiothreitol (DTT), 1M5 ml

    SDS-PAGE Sample Buffer1 ml

    Instruction Manual

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  • Introduction:The IMPACT-TWIN (Intein Mediated Purification with an Affinity Chitin-binding Tag-Two Intein) system is a novel protein purification system which utilizes the inducibleself-cleavage activity of protein splicing elements (termed inteins) to separate thetarget protein from the affinity tag. Unlike other purification systems, IMPACT-TWIN isable to isolate native recombinant proteins possessing an N-terminal cysteine and/orpossessing a reactive C-terminal thioester in a single chromatographic step withoutthe use of exogenous proteases. These reactive groups can be used in Intein-mediated Protein Ligation (IPL, Appendix II) to specifically attach proteins, peptides,or labels to the N-and/or C-terminus of a target protein (1-9) using chemistrydescribed previously (10,11). Furthermore, an intramolecular reaction makes itpossible to generate circular protein species.

    The IMPACT-TWIN system and its companion product, the IMPACT-CN proteinpurification system, were the direct result of New England Biolabs investigation intothe mechanism of protein splicing (Appendix IV, Refs. 12,13). The IMPACT-TWINsystem allows a target protein to be sandwiched between two self-cleaving inteins(Figure 9, Ref. 3). Chitin binding domains (14) present on both inteins allow theaffinity purification of the precursor protein on a chitin resin. Intein1 is a mini-inteinderived from the Synechocystis sp dnaB gene (15) engineered to undergo pH andtemperature dependent cleavage at its C-terminus (16). Cleavage of this intein canliberate an N-terminal amino acid residue other than Met on a target protein. A proteinwith an N-terminal cysteine residue can be used in IPL reactions. Intein2 is either amini-intein from the Mycobacterium xenopi gyrA gene (17) (pTWIN1) or from theMethanobacterium thermoautotrophicum rir1 gene (18) (pTWIN2). These inteins havebeen modified to undergo thiol-induced cleavage at their N-terminus (1,2,19). The useof thiol reagents such as 2-mercaptoethanesulfonic acid (MESNA) releases a reactivethioester at the C-terminus of the target protein for use in IPL. Following cleavage ofboth inteins the target protein is eluted from the chitin resin while the inteins remainbound through the chitin binding domains.3

  • This system offers many advantages: (i) the facile isolation of native proteins withoutan affinity tag that may alter its characteristics. (ii) the isolation of proteins with an N-terminal cysteine or residue other than methionine without the use of exogenousproteases which can be costly and non-specific (the intein has not been observed tocleave unexpected sites). (iii) the purification of proteins with a C-terminal thioesterfor use in IPL reactions to insert non-coded amino acids into or label a bacteriallyexpressed protein. (iv) the generation of circular protein species (see Figure 9).

    System Components:

    Cloning Vector DNAThe pTWIN vectors are used for the cloning and expression of recombinant proteinsin E. coli. The exact vector and cloning strategy that should be employed dependsboth on the desired outcome and the properties of the target protein (see ProteinPurification Strategies). Both pTWIN1 (7375 bp) and pTWIN2 (7192 bp) contain Sap Isites which allow the gene of interest to be cloned between the intein tags without theaddition of any vector derived residues at either termini of the target gene. ThepTWIN1 and pTWIN2 vectors both use a modified Ssp DnaB intein as Intein1 anddiffer only in the identity of intein2 (see Figure 9 for the relative positions of Intein1and Intein2). pTWIN1 uses a modified Mxe GyrA intein while pTWIN2 uses a modifiedMth RIR1 intein. However, both pTWIN1 and pTWIN2 contain the same multiplecloning sites which simplifies the insertion of a target gene into both vectors todetermine the optimal expression plasmid.

    pTWIN-MBP1 can be used both as a control vector and a cloning vector. Cloning of atarget gene into the Nco I to Sac I sites in pTWIN-MBP1 adds 3 amino acids to theproteins N-terminus and 23 amino acids to its C-terminus. When additional aminoacids will not alter the behavior of the target protein this linker may increase the yieldsof circular species. In the case of the 43 kDa E. coli maltose binding protein (MBP)these extra amino acids were found to permit cyclization whereas without these linker 4

  • sequences no circular MBP was detected. Cloning into the Nco I to Xho I sites inpTWIN-MBP1 can be used if a smaller linker is desired. This results in 3 amino acidsattached to the proteins N-terminus and 3 amino acids to its C-terminus.

    The pTWIN vectors utilize a T7 promoter to provide stringent control of the fusion geneexpression (20). In the absence of IPTG, basal expression of the fusion gene issuppressed by binding of the lac repressor to the lac operator sequence immediatelydownstream of the T7 promoter. The presence of the lacI gene, which encodes lacrepressor, in the pTWIN vectors permits tight transcriptional control even in E. colistrains lacking an endogenous lacI gene. Background transcription